Our laboratory has been studying the mechanism of action of the 70 kDa class of heat shock proteins (Hsp70s), which have been termed molecular chaperones because they are involved in the folding and unfolding of proteins and in the formation and dissociation of protein complexes. In these studies we have concentrated on exploring the role of Hsp70 in endocytosis in particular its ability to uncoat clathrin-coated vesicles. In many of their activities the Hsp70s require cofactors known as J-domain proteins that induce protein substrates to bind to Hsp70, and we previously discovered that uncoating also requires a J-domain protein, the minor 100 kDa clathrin assembly protein (AP), auxilin. Auxilin is a nerve specific protein and we later discovered that the non-neuronal homolog of auxilin is the 150 kDa protein GAK. During the past year we have continued our investigation of whether auxilin occurs in lower organisms and, if so, whether preventing its expression causes major defects in clathrin-mediated endocytosis in vivo. We previous showed that C. elegans has a single gene for auxilin and when auxilin expression is inhibited by RNA-mediated interference, there is a marked inhibition of clathrin-mediated endocytosis which in turn causes the worms to arrest during larval development. During the past year we determined whether auxilin was expressed in S. cerevisiae. We found that the yeast genome encodes a 668-amino acid protein that we named Aux1, which has a C-terminal J-domain that shares 40% amino acid identity with the C-terminal of mammalian auxilin. Although there is little similarity in the rest of the molecule, where presumably clathrin binds, we found that Aux1 is able to polymerize mammalian clathrin into baskets. Furthermore, the Aux1 polymerized into these baskets recruits Hsp70 to bind to the baskets at pH 6 and supports Hsp70 uncoating of these baskets at pH 7. although it is not nearly as active as mammalian auxilin. When we then deleted the Aux1 gene, we found that the resulting haploid yeast mutants showed an increase of clathrin associated with vesicles and a corresponding decrease in free clathrin in the cytosol. Furthermore, in yeast not expressing Aux1, there is a marked decrease in transport of both carboxypeptidase Y and the G-protein-coupled receptor Ste3 to the vacuole; transport of both of these proteins normally occurs through clathrin-mediated endocytosis. Finally, using immuno-electron microscopy we showed that there was more than a 5-fold increase in clathrin-coated vesicles in yeast not expressing AUX1 compared to wild-type cells. From these data, we concluded that uncoating of clathrin-coated vesicles by Hsp70 and an auxilin homolog is a fundamental step in clathrin-mediated endocytosis in yeast. During the past year we also began an investigation of the functions that Hsp70 and auxilin carry out in vivo, in particular in mammalian cells. We first asked whether dissociation and rebinding of clathrin, i.e. clathrin exchange, is a normal part of clathrin-mediated endocytosis independent of the irreversible dissociation of clathrin that occurs after vesiculation takes place. We investigated this question both in vitro using bovine brain clathrin-coated vesicles and in vivo using HeLa cells. The latter studies were carried out by tagging the clathrin light chain with GFP using the method pioneered by Keen and his associates and then measuring the rate and magnitude of the recovery of fluorescence following photo bleaching of the clathrin-coated pits at the plasma membrane. We found that, in vitro, the clathrin in clathrin-coated vesicles and clathrin baskets does not exchange with free clathrin even in the presence of Hsp70 and ATP when partial uncoating occurs. On the other hand, FRAP studies in wild-type cells showed that, after photo-bleaching a small region of the plasma membrane, there was an immediate 50-80% decrease in fluorescence intensity followed by an 80% recovery of fluorescence with a half-life of about 15 s at 37 C. Of course at least part of this recovery was due to clathrin-mediated endocytosis itself, that is the simultaneous invagination of bleached clathrin-coated pits and formation of new unbleached pits. Therefore, to determine whether clathrin exchange occurs in existing pits, FRAP measurements had to be made on clathrin-coated pits that were maintained even after clathrin-mediated endocytosis was blocked. We used two different methods to block clathrin-mediated endocytosis while still maintaining the clathrin-coated pits on the plasma membrane. First, we expressed the dynamin mutant, K44A and second, we depleted the membrane of cholesterol. Surprisingly, we found that in both cases replacement of the photo bleached clathrin occurred at about the same rate and magnitude as when endocytosis was occurring. Furthermore, by following the fate of individual clathrin-coated pits, we found that very little of this replacement was due to dissolution of bleached pits and reformation of new pits. Rather, it was caused by rapid exchange of almost all of the clathrin in the pits with free clathrin in the cytosol. Furthermore, we found that this exchange was ATP-dependent. When ATP was depleted from the cell both the rate and the magnitude of the exchange markedly decreased. Exchange also did not occur when the clathrin-coated pits at the plasma membrane were transformed into clathrin baskets just below the surface of the membrane either by potassium depletion or by treatment of the cells with hypertonic sucrose. Therefore, in agreement with our in vitro data, clathrin exchange did not appear to occur in clathrin baskets in vivo. Taken together, these data show that ATP-dependent exchange of free and bound clathrin is a fundamental property of clathrin-coated pits but not clathrin baskets, and therefore may be involved in the structural rearrangement of clathrin that occurs as clathrin-coated pits invaginate61. Jiang, R., Gao, B., Prasad, K., Greene, L.E., and Eisenberg, E.: Hsc70 chaperones clathrin and primes it to interact with vesicle membranes. J. Biol. Chem. 275:8439-8447, 2000.